J Physiol 561.

1 (2004) pp 195–203 195

Effect of timing of iron supplementation on maternal

and neonatal growth and iron status of iron-deficient
pregnant rats
L. Gambling1 , H. S. Andersen1 , A. Czopek1 , R. Wojciak2 , Z. Krejpcio2 and H. J. McArdle1
1
Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, UK
2
Department of Hygiene and Human Nutrition, Agricultural University, Wojska Polskiego 31, 60-624 Poznan, Poland

We have previously shown that maternal iron (Fe) deficiency not only reduces fetal size,
but also increases blood pressure in the offspring when they are adults. In this paper we
examine whether there are critical periods when supplementation reverses or fails to reverse
the effect both on size and on expression of genes of Fe metabolism. We made dams Fe
deficient, mated them and provided supplements of Fe in the diet from the beginning
of gestation (0.5 days), from 7.5 days or from 14.5 days. Within 12 h of birth, dams and
neonates were killed and tissues taken and examined. Fe deficiency throughout pregnancy
reduces neonatal size. Supplementation from the beginning of the first, second or third
week all reduced the effect. Maternal haematocrit was restored to normal levels only in
animals given supplements for at least 2 weeks. In contrast, the neonates’ Fe levels were
normal in all supplemented groups. These results were mirrored in liver Fe levels and in
transferrin receptor mRNA. Iron-responsive element (IRE)-regulated divalent metal
transporter 1 (DMT1) increased in maternal and neonatal liver. Non-IRE-regulated DMT1
levels did not change in the maternal liver, but decreased in the neonatal liver. H and L ferritin
mRNA levels also showed different patterns in the mother and her offspring. Finally, the
neonatal size correlated with maternal Fe stores, and not with those of the fetus. The data
demonstrate that Fe supplementation during pregnancy is most effective when given early,
rather than later, in gestation.
(Received 25 May 2004; accepted after revision 8 September 2004; first published online 9 September 2004)
Corresponding author H. J. McArdle: Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB,
UK. Email: h.mcardle@rri.sari.ac.uk

Iron (Fe) deficiency in pregnancy has serious consequences
for both the mother and her baby. In the immediate
postnatal period, these include increased risk of low
birth-weight, increased morbidity and mortality (Scholl
& Hediger, 1994; Allen, 2000; Hamalainen et al. 2003). In
the neonatal period, there is an increased risk of impaired
motor development and coordination. In children,
language development and scholastic achievement can
be affected, there are significant psychological and
behavioural effects and decreased physical activity
(reviewed byn Kapil & Bhavna, 2002; Viteri & Gonzalez,
2002). As adults, the effects persist and can result in
elevated blood pressure and cardiovascular problems
(Crowe et al. 1995; Lewis et al. 2002; Gambling et al.
2003; Lisle et al. 2003).
Iron supplementation in appropriate cases therefore
is clearly warranted. However, there is still debate
whether supplementation should be universally provided.
Additionally, neither the optimal time period nor the
dose has been agreed. For example, World Health
Organization have recommended supplementation of
about 60 mg day−1 to all women from the second
trimester onward (Stoltzfus & Dreyfuss, 1998; WHO,
2001). In contrast, Casanueva & Viteri (2003) suggest
that 60 mg day−1 in normal women is too high, and
results in haemoconcentration, decreased birth-weight
and increased prematurity. The US Institute of
Medicine (Viteri, 1998) recommends a flexible approach,
ranging from no supplementation during the first two
trimesters to 120 mg day−1 , depending on a variety of
parameters.
There is very little information about the mechanisms
underpinning the differential effects of dose and timing.
It is generally agreed that the mother uses both Fe stores
and increased absorption to supply the developing fetus.
However, how much each can or will contribute is not
known. Nor is it clear how the mother will adapt to lower
Fe status.

C The Physiological Society 2004 DOI: 10.1113/jphysiol.2004.068825
196 L. Gambling and others
In order to examine these questions, we have developed
a rat model of Fe deficiency during pregnancy. The
pups born to Fe-deficient dams are smaller than
control neonates, and have proportionately smaller
livers. However, the degree of Fe deficiency in these pups
is less than might be predicted. We have shown that
the placenta up-regulates some of the proteins of Fe
transport as a response to maternal deficiency. For
example, transferrin receptor (TfR) levels increase
markedly as deficiency increases (Gambling et al. 2001).
Iron is transferred from the transferrin receptor in the
endocytotic vesicle into the cell through a channel called
divalent metal transporter 1 (DMT1). There are at least
two isoforms of this protein. One is regulated by an
iron-responsive element (IRE) and the other is not.
The IRE form of the protein is also up-regulated in
the placenta by maternal Fe deficiency. Consequently,
the fetus accumulates Fe at the expense of its mother.
How this is reflected in fetal liver metabolism of Fe is
part of the subject of this paper. Additionally, we do not
yet know whether there is an optimum or preferable time
for supplementation. In this paper, therefore, we examine
the effect of different time periods of supplementation on
growth and development of the offspring.
Methods
Experimental diets
The experimental diets were based on a dried egg albumin
diet and conformed to American Institute of Nutrition
guidelines for laboratory animals (Williams & Mills,
1970). Iron sulphate was added to achieve levels of
added Fe of 50 (control diet), 7.5 (Fe-deficient diet) or
75 mg kg−1 (Fe-supplemented diet). Dietary ingredients
were purchased from Mayjex Ltd (Chalfont-St Peter, UK),
BDH Chemicals (Poole, UK) or Sigma (Poole, UK).
Experimental animals
Experiment was performed using 40 weanling female
rats of the Rowett Hooded Lister strain. Animals were
housed in cages under constant conditions (temperature
22◦ C; humidity 55%; 12 h:12 h light:dark illumination
photoperiod). All the animals were fed control diet
(50 mg Fe (kg diet)−1 ) for 2 weeks, before being
randomized into two groups. The first group, eight
animals, remained on the control diet throughout the
experiment, including during pregnancy, whilst the
remaining 32 animals were placed on the Fe-deficient
diet (7.5 mg Fe (kg diet)−1 ) for 4 weeks before mating.
All the rats were mated with males of the same strain.
Mating was confirmed by detection of a vaginal plug,
and this day was denoted as day 0.5. At 0.5, 7.5 and
14.5 days gestation, separate groups of eight rats were
J Physiol 561.1
taken from the deficient diet and given the supplemented
diet until parturition. The remaining eight rats continued
on the Fe-deficient diet. The pups were killed within
12 h of birth by decapitation. The dams were killed by
stunning and cervical cord dislocation. All experimental
procedures were approved and conducted in accordance
with the UK Animals (Scientific Procedures) Act 1986.
Sample collection
The number of neonates (male and female) was counted.
Maternal and neonatal blood samples were collected.
Maternal livers as well as neonatal livers, hearts, lungs,
kidneys and spleens, taken from six neonates (3 male and
3 female), chosen from each mother at random, were
rapidly dissected, weighed and frozen in liquid nitrogen
before being stored at –70◦ C.
Haematological measurements
Maternal and neonatal haematocrit were measured by
drawing blood into capillary tubes, which were then
centrifuged in a high-speed haematocrit centrifuge
(Universal 32R, Hettich; Scientific Laboratory Supplies,
Coatbridge, UK) and read in a microhaematocrit reader.
Atomic absorption spectrophotometric analyses
For determination of total Fe content in tissues, the
heat-dried liver samples were treated with nitric acid
(Ultrapure; Merck, Poole, UK). The total Fe content
in these samples was determined by graphite furnace
atomic spectrophotometry (Aanalyst 600, Perkin Elmer,
Beaconsfield, UK). Standards and quality controls were
included as appropriate.
Primers for real-time RT-PCR
Complementary DNA PCR primers for the TfR,
DMT1 IRE and non-IRE forms, ferritin H and
ferritin L were designed using Primer Express software
(version 1.5, Applied Biosystems, Warrington, UK) from
the DNA sequences GenBank Accession numbers M58040,
AF008439, AF029757, NM 012848 and XM 216824,
respectively. The primers were as follows: TfR forward
primer 1757–1779 bp, reverse primer 1818–1838 bp;
DMT1 IRE forward primer 2168–2188 bp, reverse primer
2228–2247 bp; DMT1 non-IRE form forward primer
1650–1673 bp, reverse primer 1700–1722 bp; ferritin H
forward primer 585–606 bp, reverse primer 632–656 bp;
and ferritin L forward primer 140–156 bp and reverse
primer 14–35 bp. The primer sets had a calculated
annealing temperature of 58◦ C. Primers were ordered
from MWG Biotech, Munich, Germany.


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J Physiol 561.1 Iron supplementation and fetal growth in rats 197

Table 1. Effect of duration of maternal Fe deficiency on maternal and neonatal growth

Control Deficient Suppl. day 0.5 Suppl. day 7.5 Suppl. day 14.5

Maternal weight (g) 263 ±6a 258 ± 7a 259 ± 5a 258 ± 5a 263 ± 7a

Total no. of neonates 12.9 ± 1.0a 13.1 ± 0.9a 14.4 ± 0.6a 13.3 ± 0.7a 13.6 ± 1.0a
Neonatal body weight (g) 5.40 ± 0.13a 4.92 ± 0.12b 5.32 ± 0.16ab 5.47 ± 0.17a 5.02 ± 0.21ab
Neonatal liver (ROW; g (100 g)−1 ) 4.72 ± 0.08a 3.82 ± 0.16b 4.20 ± 0.14b 4.34 ± 0.2ab 4.25 ± 0.26ab
Neonatal heart (ROW; g (100 g)−1 ) 0.46 ± 0.02a 0.55 ± 0.04b 0.50 ± 0.02ab 0.48 ± 0.02ab 0.54 ± 0.03b
Neonatal lung (ROW; g (100 g)−1 ) 1.59 ± 0.05 1.42 ± 0.03 2.00 ± 0.06a 2.08 ± 0.03a 2.12 ± 0.11a
Neonatal kidney (ROW; g (100 g)−1 ) 1.08 ± 0.01a 1.02 ± 0.04 1.09 ± 0.03a 1.07 ± 0.04a 1.20 ± 0.12a
Neonatal spleen (ROW; g (100 g)−1 ) 0.22 ± 0.01a 0.19 ± 0.05a 0.24 ± 0.03a 0.23 ± 0.02a 0.36 ± 0.20a
All results are presented as means ± S.E.M. Days indicate the time in gestation at which supplementation was started. Relative organ
weights (ROW) were calculated as a percentage of total body weight (g (100 g)−1 ). Results sharing the same superscript are not
significantly different.

DNase treatment Biosystems). Reactions were performed in 25 µl final

Total RNA was prepared by use of TRI reagent
(Helena Biosciences, Sunderland, UK) according to the
manufacturer’s instructions. To eliminate genomic DNA
contamination total RNA samples were treated with
ribonuclease (RNase)-free Dnase 1 Amplification Grade
(Invitrogen Ltd, Paisley, UK), before complementary DNA
synthesis. Ten micrograms of total RNA was added to a
total volume of 20 µl containing 2 µl 10 × reaction buffer
and 1 µl Dnase 1. The total RNA was incubated for 10 min
at 25◦ C, followed by precipitation of the RNA by adding
5 µl 3 m sodium acetate pH 4.8, 55 µl isopropanol and
30 µl diethyl pyrocarbonate (DEPC)-treated water. RNA
was precipitated at −20◦ C for at least 1 h, then centrifuged,
washed in 75% ethanol and the pellet dissolved in
DEPC-treated water. RNA concentrations were estimated
by Agilent analysis (Agilent 2100 Bioanalyser, Agilent
Technologies, Stockport, UK).
Reverse transcription
First strand complementary DNA was synthesized
by priming with hexamers using the Taqman RT
Reagent Kit (Applied Biosystems, Stockport, UK). Reverse
transcription was performed in 20 µl reactions using
200 ng of Dnase-treated RNA. Reverse transcription was
performed by addition of 12.3 µl RT mix, such that the
final concentration was 1 × buffer, 5.5 mm MgCl2 , 2 mm
dNTP, 2.5 µm hexamer primer, 8 U RNase inhibitor and
25 U MultiscribeTM RT. The mixture was incubated at
25◦ C for 10 min, incubated at 48◦ C for 30 min and heated
to 95◦ C for 5 min, and then finally cooled to 4◦ C. To
determine the presence of contaminating cDNA, reverse
transcription was omitted in the cDNA synthesis reaction.
A specific product was never detected (data not shown).
Real-time quantitative PCR
Real-time PCR amplification and analysis was performed
using a 7700 Sequence Detection System (Applied
Biosystems) and ABI prism software version 1.9 (Applied
volume with 300 nm primers and 5 µl cDNA. Magnesium
chloride, nucleotides, buffer and Taq DNA polymerase
were included in the SYBR Green Master Mix (Applied
Biosystems). The PCR amplification was performed
according to the manufacturer’s instructions and included
heating to 50◦ C for 5 min and denaturation at 95◦ C for
10 min, followed by 40 cycles with 95◦ C for 15 s and
60◦ C for 1 min. To confirm amplification specificity the
PCR products from each primer pair were subjected to
gel electrophoreses (2.5% Agarose-1000, Invitrogen, with
ethidium bromide). For all primer pairs only one product
of correct size was detected (data not shown).
Standard curves were generated from increasing
amounts of cDNA made from maternal liver control RNA.
The cycle threshold (C T ) values were used to calculate
and plot a linear regression line by plotting the logarithm
of template concentration (x axis) against the C T value
(y axis). These regression lines were used to calculate the
expression level (nanograms of total RNA) for unknown
samples.
Statistical analyses
For each dam, the data from the the pups were
averaged and recorded as a single point. One-way
ANOVA was used to determine statistical significance
between multiple data sets and verified by the least
significant difference (LSD) test. Linear regression test
was used to determine statistical significance between
continuous variables. Significance was assumed at
P = 0.05. All analyses were carried out using Statistica 5.0
(Statsoft, Polska, Krakow, Poland). The results are
presented as means ± s.e.m.
The change in expression in mRNA levels between the
control group and the treated groups is presented as a
percentage of the control, with the average of the controls
set to 100%. Each group contained amplified cDNAs
from three plates, and the average of these measurements
was used to calculate group mean and s.e.m. Significant
differences (P < 0.05) between treated and control groups

C The Physiological Society 2004
198 L. Gambling and others
Figure 1. The effect of maternal Fe deficiency on maternal haematocrit (A) and haemoglobin (B)
Animals were treated as described in the Methods and the results presented are the means ± S.E.M. of at least
6 animals per group. For dietary details see Methods section. ∗ P < 0.05; ∗∗ P < 0.01 when compared to the control
value by one-way analysis of variance.
were determined using Student’s unpaired t test, two tailed
(GraphPad Instat, version 3.05).
Results
The duration of Fe deficiency had no effect on maternal
growth, or the number of neonates. In contrast, neonatal
weight was altered by the treatment (Table 1). Those
animals born to deficient mothers were significantly
smaller, with proportionately smaller livers, lungs and
spleens, but with larger hearts (P < 0.05). The offspring
of mothers supplemented at different times were
intermediate between control and deficient offspring.
Interestingly, however, the lungs of those animals
whose mothers were supplemented were significantly
larger than either control or deficient offspring
(Table 1).
Maternal haematocrit was decreased (P < 0.05) in dams
fed the Fe-restricted diet to the end of experiment
Figure 2. The effect of maternal Fe deficiency on haematocrit of
neonatal rats
The results are the means ± S.E.M. of pups from at least 6 mothers per
group. ∗ P < 0.05; ∗∗ P < 0.01 when compared to the control value by
one-way analysis of variance.
J Physiol 561.1
compared to the control group (Fig. 1A). Similar data
were obtained for the haemoglobin levels in the maternal
blood, except that the levels were also significantly
decreased in those animals given supplements only in
the last third of pregnancy (Fig. 1B). In the neonates,
haematocrit was restored to control levels in all groups
given Fe supplements (Fig. 2). Haemoglobin levels could
not be recorded.
These responses were mirrored in the measurements
of Fe levels. In the maternal liver, Fe did not recover to
control levels unless the supplementation was given for at
least the last 2 weeks of gestation (Fig. 3A). In the neonate,
liver Fe was at control levels in all supplemented animals,
irrespective of the length of the supplementation period
(Fig. 3B).
Examination of the genes involved in Fe metabolism
in maternal and fetal liver also revealed these patterns.
Figure 4A shows TfR levels in maternal liver and Fig. 4B
shows levels in the liver of the neonates. Messenger
RNA levels are significantly elevated in the mother in
Fe-deficient animals and those supplemented for 1 week,
decreasing to control levels only in those supplemented
for 2 weeks and throughout pregnancy (Fig. 4A). In the
neonates, levels are never significantly different from
controls (Fig. 4B).
Expression of the two DMT1 transcripts, IRE regulated
and non-IRE regulated, showed different patterns
in the maternal liver. The IRE-regulated transcript
increased with increasing period of deficiency, while
the non-IRE-regulated form did not change significantly
(Fig. 5A). Different results were seen in the neonatal liver.
The non-IRE-regulated form decreased as the period
of deficiency increased, while the mRNA levels of the
IRE-regulated form increased (Fig. 5B).
We also measured levels of mRNA for H and L ferritin.
In the mothers, there were no significant changes the levels
in of either (Fig. 6A). In the neonates, levels of L ferritin


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J Physiol 561.1 Iron supplementation and fetal growth in rats 199

Figure 3. The effect of maternal Fe deficiency on liver Fe levels in the dam (A) and her neonates (B)
The results are the means ± S.E.M. of at least 6 dams or the pups from 6 dams per group. ∗ P < 0.05; ∗∗ P < 0.01
when compared to the control value by one-way analysis of variance.

mRNA did not change significantly, but H ferritin mRNA
decreased with increasing times of deficiency (Fig. 6B).
Figure 7 examines the correlation between Fe levels in
maternal and neonatal livers, and neonate birth-weight.
Interestingly, a correlation is seen between maternal liver
Fe levels and birth-weight (P < 0.0015, Fig. 7A) but not
neonatal liver Fe and birth-weight (Fig. 7B).
Discussion
The aim of this study was to determine the effect of the
timing of moderate Fe supplementation of Fe-deficient
rats during pregnancy on maternal growth, on pregnancy
outcome and on Fe status in both mothers and their
offspring. The data presented in this paper show that the
severity of Fe deficiency had no effect on maternal growth
and number of live neonates. This is in agreement with
our previous study (Gambling et al. 2002) showing that
maternal Fe deficiency does not affect viability and the
number of fetuses.
The results of the present study also confirm our
previous findings (Gambling et al. 2002) that neonatal
body weight decreases with the severity of maternal Fe
deficiency. Fetal growth restriction resulting from severe
Fe deficiency has also been demonstrated by Crowe et al.
(1995) in rats and Malhotra et al. (2002) in humans.
As might be expected, supplementation reverses the
effect. The timing is important, however. Our data show
that supplementation in the last third of pregnancy is
not as effective as it is when given earlier. Although it is
difficult to extrapolate directly from rats to humans, given
the differences in timing of developmental milestones,
the data support results published in human studies.
For example, Hamalainen et al. (2003) demonstrated

Figure 4. The effect of maternal Fe deficiency during pregnancy on TfR mRNA levels in maternal (A) and
neonatal livers (B)
Samples were taken and mRNA prepared, reverse transcribed and measured, as described in the Methods. The
results are the means ± S.E.M. of livers from at least 6 dams and 6 neonates, each taken from a different litter.
∗ P < 0.05; ∗∗ P < 0.01 when compared to the control value by Student’s unpaired t test.

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200 L. Gambling and others J Physiol 561.1

Figure 5. The effect of maternal Fe deficiency during pregnancy on DMT1 mRNA levels in maternal (A)
and neonatal livers (B)
Both the non-IRE-regulated (
) and IRE-regulated forms () were measured. Samples were taken and mRNA
prepared, reverse transcribed and measured, as described in the Methods. The results are the means ± S.E.M. of
livers from at least 6 dams and 6 offspring, each taken from a different litter. ∗ P < 0.05; ∗∗ P < 0.01 when compared
to the control value by Student’s unpaired t test.

that maternal anaemia detected in the first trimester
was associated with low-birth-weight infants, whereas the
mid- and third-trimester anaemia groups did not show
significantly different outcomes when compared with
non-anaemic women. Additionally, Cogswell et al. (2003)
have presented data suggesting that Fe supplementation,
even in women of normal Fe status, during the first
28 weeks of pregnancy, but not during the latter half, results
in a very significant increase in birth weight.
Even though the rat model has limitations, it also
provides us with the possibility of identifying which organs
are most susceptible to Fe deficiency. For example, it would
seem, from our data, that pre- and early postimplantation
embryos are most sensitive to maternal nutritional status.
These developmental events occur proportionately much
earlier in human pregnancy. Consequently, further studies
are needed to identify the developmental windows, rather
than the chronological windows, where Fe deficiency
exerts its effects.
The distribution of Fe following supplementation is
intriguing. The fact that fetal Fe levels recover before that
of the mother indicates that Fe is preferentially diverted to
the fetus as soon as it becomes available. The mechanism
for this can be deduced from this and from our previous

Figure 6. The effect of maternal Fe deficiency during pregnancy on ferritin mRNA levels in maternal (A)
and neonatal livers (B)
Both L (
) and H ferritin () were measured. Samples were taken and mRNA prepared, reverse transcribed and
measured, as described in the Methods. The results are the means ± S.E.M. of livers from at least 6 dams and
6 offspring, each taken from a different litter. ∗ P < 0.05; ∗∗ P < 0.01 when compared to the control value by
Student’s unpaired t test.

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J Physiol 561.1 Iron supplementation and fetal growth in rats 201

studies. Expression of the proteins of Fe transfer is
significantly increased in the placenta during Fe deficiency
(Gambling et al. 2002, 2003). The degree of increase
is significantly greater than that seen in the maternal
liver. Clearly, since the present study was carried out on
neonates, we have no information on the degree of changes
in TfR expression in the placenta. However, assuming
they follow the same pattern as previously described,
the Fe taken up in the supplemented animals will have
been preferentially delivered to the fetus. This is sub-
stantiated because there is no significant difference in
neonatal expression of the TfR in the liver. In the mother,
in contrast, Fe levels did not returned to normal even
following 2 weeks of supplementation.
In one sense, the results are not surprising. Iron is
critical for rapidly developing fetal and neonatal organ
systems. Iron is prioritized to haemoglobin synthesis in
red blood cells when Fe supply does not meet Fe demand.
Therefore, non-haem-containing tissues such as skeletal
muscle, heart and brain will become Fe deficient before
signs of Fe deficiency anaemia (Rao & Georgieff, 2002).
The effects on the two forms of DMT1 are also
interesting. There are data to suggest that regulation of
DMT1 by Fe is altered at different stages in gestation.
Lonnerdal and colleagues have shown that there is an
apparent developmental regulation of DMT1 in rats
(Leong et al. 2003), with greater sensitivity in the gut
occurring at postnatal day 20 rather than day 10. In one
sense, this is not surprising, since the duodenum is not
exposed to the Fe-deficient environment directly until
birth. In contrast, as we have previously shown (Gambling
et al. 2001) and confirmed in the present study and in
Lonnerdal’s paper (Leong et al. 2003), liver DMT1 levels
are increased in Fe deficiency in the pre- and early postnatal
period.
The effect on the non-IRE-regulated DMT1 has not
previously been reported. The function of this transcript
is not clear, and at present we have no obvious
explanation. Ke et al. (2003) have suggested that the two
isoforms both respond to Fe deficiency in the heart, while
Tchernitchko et al. (2002) suggest that there are other
promoters and/or regulators involved in induction of
the different isoforms. Clearly, there is much yet to be
elucidated.
The effect on ferritin mRNAs was also intriguing.
There was no significant change in the maternal liver,
or in L ferritin in the neonate, while the H ferritin
level significantly decreased both in neonates born
from Fe-deficient mothers and in those born from
mothers supplemented for 1 week only. H and L ferritin
are independently regulated proteins with both trans-
criptional and translational regulation in response to
cellular Fe level. Han et al. (2000) studied the H:L ferritin
ratio and mRNA levels in various regions of the brain
of male Fe-deficient, control and Fe-supplemented rats.
They found that ferritin H and L subunits within the
brain respond differently to Fe status. On the basis
of experiments with synthetic ferritin heteropolymers,
Levi et al. (1994) concluded that the ferritins with high
L:H chain ratios are the most efficient in incorporating
Fe (Levenson & Fitch, 2000). The data we have
obtained in this study would seem to support that
observation.
What is most surprising is that there is no correlation
between neonatal Fe levels and neonatal size. Instead,
the relationship appears to between maternal Fe and
the neonate. This suggests that the mother rather than
factors deriving from the fetus determines size at birth.
There are other data to support these observations. In
sheep, Wallace et al. (2002) have shown that placental

Figure 7. Maternal and not neonatal liver Fe levels are related to neonatal birthweight
A, the liver Fe level of each mother is related to mean litter weight. Statistical analysis was carried out by linear
regression. The dotted lines represent the 95% confidence limits. The slope of the line is significantly different
from 0 and the data are correlated at P = 0.0015. B, mean litter liver Fe levels were measured and data correlated
to mean litter weight as shown. The slope of the line is not significantly different from 0.

C The Physiological Society 2004
202 L. Gambling and others
growth is regulated by maternal nutrition and this, in turn,
regulates fetal growth. Various hormone treatments, such
as parathyroid hormone-related protein in rats (Wlodek
et al. 2004), dexamethasone in sheep (Kerzner et al. 2002)
or leptin in sheep (Thomas et al. 2001) have all been
shown to alter fetal growth (see Reynolds et al. 2004 for
a comprehensive review). At this stage, we are not certain
which hormone mediates the effect in anaemic rats, but
it is clear that the data may have considerable relevance
in understanding the relationship between maternal
anaemia and pregnancy outcome in humans.
In summary, therefore, our data show that the
developing fetus is very sensitive to maternal Fe status,
and that the period of supplementation is critical in
reversing the effects of maternal anaemia. Anaemia during
pregnancy is endemic in many parts of the world and
is a significant concern even in developed countries. It
is well established that supplementation is of limited
value outside of well-controlled experimental studies.
Our data may provide an explanation for this, in that
if it is given at an inappropriate time, it will be
less effective at reversing the consequences of maternal
anaemia. Certainly the data would suggest that early
supplementation will be more efficacious than during
the last trimester. The mechanisms underlying these
observations clearly remain to be determined, but a better
understanding will be very valuable in developing the
appropriate strategies for treatment of mothers suffering
from anaemia during pregnancy.
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Acknowledgements
This work was supported by the European Union
(QLK1-1999-00337), The International Copper Association
and Scottish Executive Environmental and Rural Affairs
Department. We are grateful for the invaluable assistance of
the staff of the Biological Resources Unit and Lynne Beattie for
technical assistance, and to Ann White for proofreading and
secretarial assistance.